A rise in the cytosolic free Ca2+ concentration, [Ca2+]c, is an important second messenger in many types of cells. In arterial myocytes, an increase in [Ca2+]c is known to signal changes in the growth state of cells and is the immediate trigger for contraction. Ca2+ can enter the cytosol from the extracellular fluid through voltage-gated and/or receptor-operated channels. In addition, Ca2+ is released from internal stores in the sarcoplasmic reticulum (SR). For arterial myocytes, however, there is little quantitative information available regarding intracellular Ca2+ concentrations, the spatial distribution of that Ca2+, or the changes that are evoked during activation by vasoconstrictors such as vasopressin and serotonin. The goal of this research program is to address those issues and, thereby, gain an understanding of some of the factors that regulate the distribution of intracellular Ca2+ in arterial myocytes. The emphasis is on the relative roles of the SR and the sarcolemma in regulating the cellular response during agonist-induced activation. Digital analysis of images of fura-2 fluorescence will be used to spatially resolve intracellular [Ca2+] in single smooth muscle cells from cultured rat aorta and mesenteric artery. Three fura-2 loading procedures (fura-2/AM incubation, free fura-2 loading by ATP-induced transient membrane permeablization, and fura-2/AM incubation with subsequent cytosolic dye depletion) will be used to resolve separate pools of intracellular Ca2+ (e.g. SR & cytosol). In addition, co-localization of fura-2 and organelle specific fluorescent probes (e.g. rhodamine 123) will be used to identify the location of fura-2 within organelles. In this way Ca2+ stores mobilized by caffeine, ryanodine and IP3 will be spatially resolved. The sources of Ca2+ for agonist-induced Ca2+ transients will be studied. Caffeine and ryanodine will be used to pharmacologically separate the SR component of agonist-induced changes in intracellular Ca2+ from the contribution made by an influx of extracellular Ca2+ into the cytosol. Ca2+ channel antagonists and zero Ca2+ media will be used to assess the importance of an influx of extracellular Ca2+ in mediating agonist-induced release of Ca2+ from the SR. The role of cytosolic Ca2+ in modulating IP3- mediated Ca2+ release from SR will also be explored. The results of these experiments should provide new information about the regulation of Ca2+ in arterial myocytes. Moreover, these findings may be useful in elucidating pathophysiological processes such as those associated with hypertension.